5 replies to this topic

[SarahLee8]

Hello, I'm looking for some help in regards to the design of bisulfite sequencing primers.

As I'm reviewing the literature, I've seen that some papers refer to primer sets designed specificially for either the bottom strand or the top strand.  This may have a simple explanation that I am missing, but I am still having a difficult time grasping this... When we design primers for normal PCR, we pick a primer from the top strand and a primer from the reverse strand.  How would we pick primers from just one strand if the sequences are the same, but just the reverse complement of each other?  I appreciate any insight you can provide!

Thank you.

[FMK]

Could you please cite which papers you refering to?

[pcrman]

After bisulfite modification, two DNA strands are no longer complementary. When design bisulfite PCR primers, we usually design them on the sense DNA strand, though it is not wrong to design primers on the minus strand. This is based on the fact that DNA methylation is symmetric. I hope I have made myself understood.

[prabhubct]

Because the two strands of DNA are no longer complementary after bisulfite modification, strand-specific primers are used for PCR amplification. Usually the sense strand is chosen for primer design.

http://www.protocol-...d-PCR-3161.html

Edited by prabhubct, 18 September 2012 - 09:58 AM.

[SarahLee8]

Thank you very much for all of your replies.  The paper I'm specifically looking at is:

Hansen, R. S. "X Inactivation-specific Methylation of LINE-1 Elements by DNMT3B: Implications for the Lyon Repeat Hypothesis." Human Molecular Genetics 12.19 (2003): 2559-567.

[mniku]

Here's another confused beginner... I'm still not 100% sure I understand the talk about "strand-specific" primers. Isn't this just like "traditional" PCR primer design, except that one of course uses a bisulfate converted sequence?

I mean, I usually use just the sense strand sequence as the design template and use something like Primer3 to pick the primers. If I now use the bisulfate-converted sense sequence, I will obviously get a reverse (antisense) primer that is fully complementary to the sense strand, and a forward (sense) primer that is NOT complementary to the antisense strand because of the bisulfate conversion. So during the first round of the PCR, only the sense strand will be amplified. And after that, the forward primer will bind to the new products and the rest is exponential amplification of the original sense strand. Is this what the strand specificity means?

I just don't see what else could I do. I mean, if I did design the primers fitting to both of the converted strands, they would amplify the original templates all right, but not the products, so the amplification would be only linear.